Mycobacterium tuberculosis (M. tuberculosis) remains one of the world's most important pathogens, with a mortality rate exceeding 1.5 million deaths annually (Dye C, et al. (2013) Annu Rev Public Health. 34:271-286). Despite study of this pathogen for more than a century, the spectrum of natural lipids within M. tuberculosis membranes is not yet fully defined. For example, the products of many genes annotated as lipid synthases remain unknown (Camus J C, et al. (2002) Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv Microbiology 148:2967-2973), and mass spectrometry detects hundreds of ions that do not correspond to known lipids in the MycoMass database (Layre E, et al. (2011) A comparative lipidomics platform for chemotaxonomic analysis of Mycobacterium tuberculosis, Chem Biol 18(12):1537-1549 provides a method to detect individual lipids that are present in infectious bacteria that cause tuberculosis disease (M. tuberculosis) versus attenuated bacteria that are used in vaccines such as BCG. In general, it is important to distinguish patients with tuberculosis from those vaccinated with BCG because the treatments are different and more than 1 billion people have been vaccinated with BCG.
Methods of detecting the presence or absence of the bacteria typically include culturing a sample suspected of having bacteria. However these tests may take over two weeks to complete depending on how long it takes to isolate and grow the bacteria. Accordingly, while such biochemical testing is relatively inexpensive, it is time consuming to grow and subculture bacteria in a sample to reach the minimal concentration of bacteria needed for testing. One standard for the diagnosis of active pulmonary tuberculosis is sputum smear microscopy for acid-fast bacilli. If a patient's sputum tests positive for M. tuberculosis they have active pulmonary tuberculosis, are considered highly infectious, and are placed on an exhaustive drug regimen for treatment. However, sputum smear microscopy has low sensitivity and it is estimated that sputum smear microscopy at best detects 25-60% of people with active pulmonary tuberculosis. The method also has relatively poor limits of detection as it requires the presence of at least 10,000 MTb bacilli/mL. An alternative to culture positivity is to detect bacterial DNA by PCR, but such methods are expensive and difficult to use in resource limited settings in which the tuberculosis epidemic is prevalent.
Serologic tests exist for M. tuberculosis diagnostics, but they continue to undergo development and tend to be more specific for exposure than active disease. Some commercialized tests use immunodominant antigens to detect immunoglobulin classes (like IgG) in an ELISA or dipstick format. Serological tests are estimated to detect one-third to three-quarters of sputum smear-positive cases of MTb. They detect a significantly smaller portion of smear-negative cases with HIV co-infection. In fact, for people infected with both HIV and MTb, serological tests detect less than one third of patients with the active form of the disease. Many molecular targets of current serological tests, such as mycolic acid and lipoarabinomannan, are produced by mycobacterial in the environment or vaccine strains. It is thought that vaccination or exposure to environmental mycobacteria causes false positive test results in patients with no M. tuberculosis infection. Identification of molecular targets that are expressed solely or mainly by M. tuberculosis and not other common mycobacteria is therefore expected to yield fewer false positive tests.
A widely used test to determine M. tuberculosis (TB) is the PPD (purified protein derivative) skin test. Patients are administered a small shot that contains PPD under the top layer of the skin. A bump or small welt will form, which usually goes away in a few hours. If the area of skin that received PPD is still reactive 48 to 72 hours after the injection, the test results are positive. People who received a BCG (bacille Calmette-Guerin) vaccine against tuberculosis give a false-positive reaction to the PPD test. Many foreign-born people have had the BCG vaccine, though it is not given in the U.S. due to its questionable effectiveness. Accordingly, even if one has been vaccinated, they could still carry the disease. The PPD test does not discriminate between BCG vaccines and patients with M. tuberculosis infection and tuberculosis disease. Thus, diagnosis of M. tuberculosis infection is complicated by the fact that approximately 1 billion people worldwide have been treated with live Mycobacterium bovis Bacille Calmette Guerin (BCG) bacteria as a vaccine, and those persons that have been treated with this vaccine will show a false positive reading in diagnostic tests. In addition, the PPD test also known to show a positive reaction when a subject is infected with non-tuberculosis mycobacteria.
Accordingly, more efficient methods and systems are needed to screen patients suspected of having M. tuberculosis. In particular, identification of molecules that are produced by M. tuberculosis but not BCG provides the opportunity to develop molecular targets that will not cause false positive serological tests or biochemical tests that directly detect the molecule of interest in ELISA or related methods.
Approximately 1.7 billion are infected with M. tuberculosis worldwide. A test that can distinguish people that have been treated with the common BCG vaccine, or that have non-tuberculous mycobacteria, from people that actually have the pathognenic M. tuberculosis is of great value.